Search Results (4)

Adeno-associated virus (AAV) vectors are the preferred gene delivery tool in gene therapy owing to their safety, long-term gene expression, broad tissue tropism, and low immunogenicity. Affinity ligands that can bind multiple AAV serotypes endure harsh clean-in-place (CIP) conditions and are critical for industrial-scale purification. However, current ligands lack broad serotype recognition and adequate alkaline stability, which limits their reusability in large-scale manufacturing. In this study, we employed a competitive biopanning strategy to isolate a single-domain antibody (VHH) that simultaneously binds AAV2, AAV8, and AAV9. The VHH retained structural integrity and binding activity after exposure to 0.1 M NaOH, demonstrating robust alkaline stability. Structural modeling revealed that the VHH primarily recognizes the DE loop region of the VP3 capsid protein across the three serotypes, explaining its cross-serotype reactivity. Affinity chromatography using the VHH yielded infectious AAV particles, confirming its potential for downstream processing. This strategy provides a versatile platform for developing high-performance AAV affinity ligands and may be extended to other viral vector systems. Full article

Background: Since the emergence of SARS-CoV-2 in the human population, the virus genome has undergone numerous mutations, enabling it to enhance transmissibility and evade acquired immunity. As a result of these mutations, most monoclonal neutralizing antibodies have lost their efficacy, as they are unable to neutralize new variants. Antibodies that neutralize a broad range of SARS-CoV-2 variants are of significant value in combating both current and potential future variants, making the identification and development of such antibodies an ongoing critical goal. This study discusses the strategy of using heterologous antigens in biopanning rounds. Methods: After four rounds of biopanning, nanobody variants were selected from a phage display library. Immunochemical methods were used to evaluate their specificity to the S protein of various SARS-CoV-2 variants, as well as to determine their competitive ability against ACE2. Viral neutralization activity was analyzed. A three-dimensional model of nanobody interaction with RBD was constructed. Results: Four nanobodies were obtained that specifically bind to the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein and exhibit neutralizing activity against various SARS-CoV-2 strains. Conclusions: The study demonstrates that performing several rounds of biopanning with heterologous antigens allows the selection of nanobodies with a broad reactivity spectrum. However, the fourth round of biopanning does not lead to the identification of nanobodies with improved characteristics. Full article

CD117 (c-Kit) is a tyrosine kinase receptor that is overexpressed in multiple dog tumors. There is 100% homology between the juxtamembrane domain of human and canine CD117, and many cancer-causing mutations occur in this region in both species. Thus, CD117 is an important target for cancer treatment in dogs and for comparative oncology studies. Currently, there is no monoclonal antibody (mAb) specifically designed to target the exposed region of canine CD117, although there exist some with species cross-reactivity. We panned a naïve phage display library to isolate antibodies against recombinant CD117 on whole cells. Several mAbs were isolated and were shown to bind recombinant canine CD117 at low- to sub-nanomolar affinity. Additionally, binding to native canine CD117 was confirmed by immunohistochemistry and by flow cytometry. Competitive binding assays also identified mAbs that competed with the CD117 receptor-specific ligand, the stem cell factor (SCF). These results show the ability of our cell-based biopanning strategy to isolate a panel of antibodies that have varied characteristics when used in different binding assays. These in vitro/ex vivo assessments suggest that some of the isolated mAbs might be promising candidates for targeting overexpressed CD117 in canine cancers for different useful applications. Full article

Traditional, horse-derived antivenin is currently the most efficient treatment against snake bites. However, it is costly and has unpredictable side effects. Thus, alternative, cost-effective strategies for producing antivenin are needed. In this study, we immunized hens with inactivated NNA venom proteins from the cobra Naja naja atra (NNA). Purified yolk IgY antibodies showed specific anti-NNA binding activity comparable to that of the equine-derived antivenin. We used phage display technology to generate two antibody libraries containing 9.0 × 10⁸ and 8.4 × 10⁸ clones with a short or long linker, respectively. The phage ELISA indicated that anti-NNA clones displaying single-chain variable fragments (scFv) were significantly enriched after biopanning. The nucleotide sequences of the light and heavy chain genes of 30 monoclonal scFv antibodies were determined and classified into six groups with the short linker and nine groups with the long linker. These scFv clones specifically bound to NNA proteins but not to venom proteins from other snakes. Their binding affinities were further determined by competitive ELISA. Animal model studies showed that anti-NNA IgY antibodies exhibited complete protective effects, while a combination of scFv antibodies raised the survival rates and times of mice challenged with lethal doses of NNA venom proteins. Full article